Cloning And Identification Of Meloidogyne Javanica Gene Mj-4D01

Meloidogyne javanica is one of the most improtant root-knot nematode in the Southern China.The hosts of M.javanica is up to 2000 and it causes imcomputable losses in agriculture of China every year.During the parasitism,M.javanica would inject numerous proteins which secreted from esophageal glands into plant cells,similar to other root knot nematodes.These proteins,as known as effectors,play important role in the process of infection,like degrading the cell walls,surpressing the immunity system,inducing the formation of feeding sites.In order to understand the molecular mechanism of M.javanica pathogenicity,provide a theoretical basis for the control of M.javanica,the identifying and analyzing the function of effectors are necessary.In this paper,one of the potential effector,Mj-4D01,from M.javanica was cloned.Brief analysis of this effector were completed by in situ hybridization,in vitro RNAi,q RT-PCR and yeast two-hybrid methods.The results are as follows:1.The Mj-4D01 gene was cloned by the method of homology-based cloning.The full length of c DNA is 525 bp,and the DNA is 1012 bp which contains four introns.This gene had the highest homology with Mi-MSP3(97%)from Meloidogyne icognita.The ubiquitous homologous proteins of this protein were found by BLAST contrast in root knot nematodes.Mj-4D01 codes 174 amino acid and the front 24 amino acid is signal peptide.A specific domain was found at the C-terminal which called ground-like domain.2.We use a Mj-4D01 specific probe labeled by Digoxin to find a localization by in situ hybridization.The results show that Mj-4D01 is localized in subventral esophageal at pre-j 2 of M.javanica.3.q RT-PCR showed that the expression level of Mj-4D01 was very low in the egg phase and pre-J2 of M.javanica,increased obviously in J3(18 dai),then decreased significantly after this stage,expressions in mature female stages are also low.It showed that Mj-4D01 plays a specific role in the J3.4.In the in vitro RNAi,the expression of Mj-4D01 was obvious down compared to control group after soaking in ds RNA.The number of mature female and total amounts of nematodes had a significant decline after 35 dai.5.A vector for transient expression was constructed for subcellular localization,the Mj-4D01 was fused with GFP and drived by 35 s promoter,and transient expression was performed in the tobacco leaves.The results show that Mj-4D01 was accumulated in the plastids.6.In yeast two hybrid system,we screened 4 portential protein from c DNA library of Arabidopsis thaliana.After contransformation,we futher comformed a protein,Rieske iron-sulfur protein precursor,which is related to defense response in plant.Above all,Mj-4D01 is a secreted effectors from M.javanica and play a role in parasitism,and it has potential to regulate the immunity system in plant.